A. Field of the Invention
The present methods are directed to the formation of multi-layered, microcapsules containing a variety of compounds, including pharmaceuticals. The present methods rely on controlling fluid shear forces in the microcapsule forming solutions. This low-shear approach to microcapsule formation yields more spherical microcapsules and a desirable size distribution. Methods are also provided for coating microcapsules with polymeric coatings. One such method involves the use of electrostatic fields to facilitate coating microcapsules with polyvinyl pyrrolidone.
B. Description of the Related Art
Many drug and enzyme therapeutics cannot be injected intravenously. Others can be injected, but rapidly degrade before reaching the target tissue. Some drugs and enzymes are cleared from the blood by the liver or kidneys so quickly that their biological half-life is too short to be of therapeutic value. Still other drugs are insoluble in aqueous solutions. Since intravenous injection in hydrocarbon solvents is not well tolerated by patients, such drugs are difficult to administer.
One method for overcoming these limitations is encapsulation into microcapsules or liposomes. Encapsulation of therapeutics can enable delivery to target organs where they can be released. Incorporation of therapeutics into microcapsules facilitates delivery by parenteral injection, nasal inhalation and dermal administration and provides for sustained drug release.
The size and shape of the microcapsules is critical for the distribution and drug delivery in the tissues. Typically, microcapsules of 1-20 micron diameter are ideal for intravenous administration, whereas, 50-300 micron diameter microcapsules are used for intraarterial delivery and 300 micron or greater for intraperitoneal administration. In each size range, highly uniform microspheres are needed for maximum packing densities and maximum drug payload delivery to target organs or tumors.
Microcapsules, such as liposomes, can be formed from amphiphilic molecules, such as phospholipids, that are capable of assembling into bilayers when dispersed in aqueous solutions at concentrations at or above their critical micelle concentrations. Typically, in liposomes that carry pharmaceuticals, the pharmaceutical is dissolved in the aqueous phase. However, drugs of limited solubility in aqueous solvents are difficult to incorporate into liposomes. Lipophilic drugs in liposomal formulations are only carried, if at all, in the hydrophobic region of the lipid bilayer. Some drugs are so insoluble that they do not associate with the bilayer and, therefore, have very low encapsulation efficiencies. Certain liposomal drug formulations, including anti-tumor liposomes containing doxorubicin [Gabizion et al. 1992] or muramyl tripeptide have been studied extensively in clinical trials.
Other methods of forming microcapsules are based on liquid-liquid dispersions of aqueous drugs and organic solvents. The dispersion methods often require emulsification of the aqueous phase into organic carrier solutions by shear, bubbling or sonication. These methods typically produce water-in-oil (WIO) type liposomes, for which a second requisite step is the removal of the organic solvent (typically by evaporation) to form reverse-phase evaporation vesicles or stable plurilamellar vesicles. These vesicles are rarely spherical and their size distribution is quite heterogeneous. Typically, in order to generate multilamellar vesicles, film casting with organic solvents, hydration and sizing using filtration through inert membrane filters is required [Talsma and Crommelin 1992]. Sophisticated, multi-step emulsion technology is required and yields of uniform type and size are often very low.
Liquid microemulsions also have been developed as drug delivery systems, especially for drugs that are poorly soluble in aqueous carriers. A microemulsion typically contains droplets in the range of 0.1 to 1 micron in diameter. Such microemulsions are characterized by very fluid and dynamic micelles which are formed by sequential mixing one immiscible phase with another using surfactants and co-surfactants [Bhargava et al. 1987]. Typically, surfactants that produce water-in-oil (W/O) microemulsions have a hydrophilic-lipophilic balance (HLB) rating of 3 to 6, while those that produce oil-in-water (O/W) microemulsions have an HLB of 8 to 18. The surfactants can be non-ionic, ionic, or amphoteric. Often, medium chain-length alcohols are added as the co-surfactant in the last step in achieving the final microemulsion.
The major disadvantages of microemulsions is that each micelle (liquid capsule) is too small (typically, less than 1.0 micron) for deposition in larger vascular beds when administered by intravascular injection. Therefore, microemulsions are not suitable for chemoembolization type treatment of vascularized tumors.
Additionally, since microemulsions are true colloidal suspensions, they cannot be scaled up to large enough size for many intravascular drug delivery applications. Microemulsions formed with lipid soluble anti-tumor agents and low-density lipoproteins (LDLS) have been used to target drugs to neoplastic cells that require large amounts of cholesterol for synthesis of cell membranes [Halbert et al. 1984]. However, LDLs also attract phagocytes making the amount of drug actually delivered to the tumors and thence the therapeutic dose difficult to determine.
Solid matrix microspheres may be also used for transporting adsorbed drugs within the matrix. For instance, U.S. Pat. No. 4,492,720 to Mosier disclosed methods for making microspheres to deliver chemotherapeutic drugs (including Cis-Platinum) to vascularized tumors. This method of preparing microspheres is accomplished by liquid encapsulation and solid-phase entrapment wherein the water-soluble drug is dispersed in a solid matrix material. The method involves dissolving the aqueous drug and the matrix material in an organic solvent, in which they are mutually soluble, then dispersing this mixture in a second organic solvent to form an emulsion that is stable enough for intravascular injection.
Other solid-matrix approaches have utilized copolymers such as polyvinyl chloride/acrylonitrile dissolved initially in organic solvents to form microparticles containing aqueous enzyme solutions. U.S. Pat. No. 3,639,306 to Sternberg et al. discloses a method of making anisotropic polymer particles having a sponge-like inner support structure comprising large and small void spaces and an outer, microporous polymer film barrier. A multiple-step batch process is used which entails removal of the organic solvents used to dissolve the polymers prior to addition of aqueous components.
Solid-matrix microspheres, however, are often not perfect spheres thereby limiting the packing density. Additionally, many drugs cannot be trapped or adsorbed in these systems at effective concentrations and drug-release rates are often not constant.
Density-driven phase separation and stratification into horizontal layers of the immiscible solutions used to form microcapsules presents a major difficulty in the commercial preparation of microcapsules. This problem limits microcapsule yields and leads to the creation of irregularly shaped capsules of variable sizes. Non-uniformity in capsule preparations limits microcapsule packing density thereby limiting the quantities of drug that can be delivered to target sites. Even when microcapsules are formed, these forces destabilize them in some cases causing them to burst.
These problems have been overcome to a limited extent through the use of multi-step, batch processes that include mechanical mixing and solvent evaporation steps [Talsma and Crommelin 1992]. However, each batch step suffers losses that reduce overall efficiencies.
Conventional methods do not permit simultaneous formation of the outer skin as the microcapsule itself is formed. Many conventional therapeutic microspheres have natural phospholipid outer skins (usually in combination with cholesterol and a fatty amine) and therefore are subject to elimination by immune cells. Other conventional methods use sialic acid and other coatings on the lipid bilayer to mask the liposomes from detection by the scavenging systems of the body. Without an adequate outer skin, microcapsules often coalesce thereby reducing shelf life.
For instance, U.S. Pat. No. 4,855,090 to Wallach, discloses a method of making a multilamellar lipid vesicle by blending an aqueous phase and a lipophilic phase using a high shear producing apparatus. The lipophilic phase is maintained at a high temperature (above the melting point of the lipid components) and is combined with an excess of the aqueous phase, which is also maintained at a high temperature. U.S. Pat. No. 5,032,457 to Wallach discloses a paucilamellar lipid vesicle and method of making paucilamellar lipid vesicles (PLV). The method comprises combining a nonaqueous lipophilic phase with an aqueous phase at high temperatures and high shear mixing conditions, wherein the PLVs are rapidly formed in a single step process.
U.S. Pat. No. 4,501,728 to Geho et al. discloses the encapsulation of one or more drugs or other substances within a liposome covered with a sialic acid residue for masking the surface of the membrane from scavenging cells of the body utilizing techniques known for the production of liposomes. In one embodiment, additional tissue specific constituents are added to the surface of the liposome, which cause attractions to specific tissues. Similarly, U.S. Pat. No. 5,013,556 to Woodle et al. provided methods for making liposomes with enhanced circulation times. Liposomes created by this method contain 1-20 mole % of an amphipathic lipid derivatized with a polyalkylether (such as phosphatidyl ethanolamine derivatized with polyethyleneglycol). U.S. Pat. No. 5,225,212 to Martin et al. discloses a liposome composition for extended release of a therapeutic compound into the bloodstream, the liposomes being composed of vesicle-forming lipids derivatized with a hydrophilic polymer, wherein the liposome composition is used for extending the period of release of a therapeutic compound such as a polypeptide, injected within the body. Formulations of "stealth" liposomes have been made with lipids that are less detectable by immune cells in an attempt to avoid phagocytosis [Allen et al. 1992]. Still other modifications of lipids (i.e., neutral glycolipids) may be affected in order to produce anti-viral formulations (U.S. Pat. No. 5,192,551 to Willoughby et al. 1993). However, new types of liposomes and microcapsules are needed to exploit the various unique applications of this type of drug delivery.
U.S. Pat. No. 4,201,691 to Asher et al. discloses a method for forming microcapsules from immiscible liquids wherein one fluid is forced under pressure through a porous membrane into a second fluid to form a dispersion of small bubble-like microdroplets. The pressure drop across the porous membrane determines the amount and size of the microdroplets or microcapsules in the resulting dispersion. The dispersion is forced through a porous fluid dispersing layer into the outlets of a nonporous material and exits the outlets as microcapsules. The microcapsule size distribution in these preparations is limited by the immutable physical characteristics of the porous fluid dispersing layer containing the multiphase dispersion just prior to its passage through the outlets. The Asher process is limited to the production of two layered microcapsules and it does not allow for coating or electrodeposition of additional coatings of microcapsules prior to transfer to another vessel.
Microencapsulation of liquid droplets has been accomplished by forcing aqueous solutions through a nozzle to create an aerosol within a cloud chamber containing an encapsulating material such as wax in vapor phase. Microcapsules are formed by passage of the liquid microdroplet through a coating vapor. In these systems the formation and curing of the outer capsule layer must occur before the microcapsules reach the walls of the cloud chamber. In a variation of this method, an electrostatic field is introduced into the cloud chamber. The field causes an electrostatic attraction between the coating material and surface of the oppositely charged liquid microdroplet. Yamati et al., Illinois. Inst. Of Technology Research Institute (IITRI) 1982; "Microencapsulation in Space (MIS)" on Shuttle Flight STS-53, Dec. 1992.
Microcapsules formed in cloud chambers are limited to diameters of less than 1 micron because of free fall dynamics that tend to break up larger microdroplets. Moreover, the formation time of microcapsules in cloud chambers is limited to the residence time of the droplet in its trajectory through the chamber. If the shell does not form before the droplet collides with the chamber wall, the microcapsule is destroyed. The fluids that can be used to form microdroplets also limit the technique. Only those fluids with suitable aerosol forming characteristics can be used and those fluids must be charged or they cannot be coated by the oppositely charged coating materials in the chamber. Furthermore, the process can only be used to create microcapsules having two layers and it does not allow for immediate coating or electrodeposition with additional charged coatings.
It is known that microgravity can be advantageously utilized to induce and maintain crystal growth due to the lack of density driven convective flow in liquids. U.S. Pat. No. 4,909,933 to Carter et al. discloses an apparatus for carrying out crystallization of proteins and chemical syntheses by liquid-liquid diffusion in microgravity environments. The apparatus comprises a housing having a plurality of chambers and a valve which separates at least two of the chambers so as to allow controlled fluid flow.
The disadvantages of conventional liposomes or microcapsules include manufacturing methods that require many batch process steps to: 1) form the liposomes, 2) remove unwanted organic solvents, detergents, and 3) harvest the proper size and shape microparticles for optimum pharmacologic efficacy [Talsma and Crommelin 1992]. Also conventional liposomes often use natural lipids and lecithins (from eggs, soybeans and other inexpensive sources) which attract phagocytic immune cells that rapidly remove the liposomes from the circulatory system before they arrive at the target tissue. This creates variable dose-responses making calculations of pharmacokinetics and therapeutic doses very difficult [Allen 1988]. Major difficulties with commercial preparation of microcapsules often involves density-driven phase separation of the immiscible carrier fluids, especially when forming water/oil systems.
These drawbacks limit the yield, make it difficult to harvest the proper size particle, result in microparticles that are not spherical nor uniform in size, thereby limiting the packing density (and drug payload delivered) when the microcapsules arrive at the arterioles or capillaries in the target issues. Liposomes have a bilayer outer membrane which requires that the entrapped drug must be soluble in both the aqueous and lipid phases in order to outwardly diffuse. This limits the type of drugs that can be released from conventional liposomes and the mole ratio of aqueous to lipid phases limits the amount of lipid drug that can be delivered.
New methods are needed for forming spherical multilamellar microcapsules having alternating hydrophilic and hydrophobic liquid layers, surrounded by flexible, semi-permeable hydrophobic or hydrophilic outer membranes which can be tailored specifically to control the diffusion rate. In particular, methods of making such microcapsules are needed which do not rely on batch processes involving, mechanical mixing and/or solvent evaporation. Moreover, there is clearly a need for methods and compositions that allow for uniform size and more spherical microcapsules. Methods are needed for controlling microcapsule formation such that microcapsules of defined diameters can be produced in a single apparatus independently of an immutable fluid dispersing layer. These methods should also be capable of producing microcapsules having a plurality of shells around their cores and therefore allow coating microcapsules with protective polymeric shells. New methods of coating microcapsules with polymeric shells are needed so that microcapsules having diameters greater than 1 micron can be coated. Microcapsules produced by such improved methods would be particularly useful in the delivery of pharmaceutical compositions.